The long-term objective of this research is to understand how bacterial cells maintain metal ion homeostasis. Essential metal ions must be obtained from the environment, transported into the cell, and trafficked to the correct intracellular destinations. When metal ions are limiting, high affinity uptake mechanisms are induced. When in excess, the cell induces the synthesis of efflux, detoxication, or storage mechanisms. The ability to obtain sufficient metal ions from the host is crucial to bacterial pathogens, metal uptake systems are highly expressed in vivo, and surface-exposed transport proteins are often targets for the host immune response. Disorders of metal ion homeostasis are also a major cause of human disease including anemias, Menkes', and Wilson's diseases, and several other neurodegenerative diseases. The genetic responses to changes in metal ion availability are coordinated by metalloregulatory proteins. In the Gram-positive bacterium Bacillus subtilis metal-specific regulators control the uptake of Mn (MntR), Fe (Fur), and Zn (Zur), and others regulate efflux mechanisms induced when metal ions are in excess (CueR, CzrA, ArsR, YdeT). We will measure the metal ion content and quotas needed for growth for both wild-type and mutants altered in metal ion homeostasis. We will test the hypothesis that adsorption of metal ions to the cell wall plays a role in both uptake and storage of metals and we will characterize pathways for Fe and Zn uptake. We will test the hypotheses that MrgA and Dps function in Fe storage and detoxification and that Zn-containing ribosomal proteins provide a major intracellular Zn store that can be mobilized under Zn limitation. Genetic and biochemical approaches will be used to further our understanding of metal-sensing by MntR, Fur, Zur, CueR, and ArsR family members by site-directed mutagenesis of metal ligands, in vivo analyses of altered function mutants, and structure-determination methods.